Several cDNA cloning strategies have been developed, however cDNA library cloning techniques rely on the selection and screening for expression of the gene of interest. Selection and screening can be time consuming and expensive. Also, traditional methods isolate partial target sequences, and the complete sequence must be pieced together from several clones or synthesized with techniques such as the Polymerase Chain Reaction.
Molecular cloning of cDNAs was first reported by Rougeon and Mach, PNAS USA 73, 3418-3422 (1976) and by Efstradiadis et al., Cell 7, 279-288 (1976). Since then, the technology has continued to be refined. Library cloning techniques are now commonplace and described in textbooks, such as, MOLECULAR CELL BIOLOGY, Darnell, Lodish and Baltimore (second edition, Scientific American Books, Inc., 1990). These techniques generally start with total RNA from cells. Generally, it is preferable to use mRNA from cells believed to synthesize the target protein. To make the complimentary DNA (cDNA), a short primer strand is hybridized to the mRNA near the 3' end. Most eukaryotic mRNA have a 3' poly(A).sup.+ tail, therefore, the primer is often poly(dT). Reverse transcriptase is then employed to add nucleotides to the primer to generate a cDNA, a DNA copy of the RNA molecule. The newly generated cDNA is called the first strand cDNA. The mRNA is then removed from the first strand cDNA, leaving the single stranded cDNA.
Next, a second strand cDNA is synthesized using the first strand cDNA as a template strand, to form a double stranded cDNA molecule. Double stranded cDNA can be made via several methods, for example, terminal transferase will add bases to a free 3' end of the single strand cDNA. If a poly(dC) tail is added to the 3' end of the first strand cDNA, then a poly(dG) primer and DNA polymerase can be employed to add nucleotides to the primer, thus creating the second strand cDNA. The second strand cDNA is annealed to the first strand cDNA and forms a complete double stranded DNA copy of the original mRNA.
The double stranded cDNA is then inserted into a plasmid vector having a marker. One method for inserting the cDNA into a plasmid vector involves cutting the plasmid vector with a restriction enzyme, and adding homopolymeric tails onto both ends of the cDNA and to both of the cut plasmid ends. Once the double stranded cDNA ends are annealed to the plasmid ends, the strands are joined by DNA ligase.
The plasmids are then transformed into an organism, such as E. coli, which lacks the marker of the plasmid vector. Cell colonies are grown and colonies having the marker are selected. For example, if the marker was ampicilin resistance and the cells were grown in the presence of ampicilin, only those cells having a plasmid conferring ampicilin resistance will grow. Then, radiolabeled DNA probe will be used to screen the cDNA library to look for the hybridized clones. Whether an inserted cDNA is present in the plasmid, and if so, if it is a partial or full-length target cDNA, is tested by sequencing and functional assays. It is common that many of the isolated and copied cDNAs, or clones, must be sequenced and analyzed before a partial or full-length target cDNA is isolated. This process of screening plasmids for a cDNA insert is expensive and time-consuming.
Okayama and Berg, Mol. Cell. Biol. 2, 161-170 (1982), transcribe mRNA into cDNA using a T-tailed cloning vector as primer. This method also requires a large amount of screening of plasmid vectors in search of vectors containing a partial or full-length target cDNA.
Gubler and Hoffman, Gene 25, 263-269 (1983), which describes synthesis of DNA in two steps by combining classical oligo(dT)-primed first-strand synthesis with RNase H-DNA polymerase I-mediated second strand synthesis. This method also requires a large amount of screening of plasmid vectors in search of vectors containing a partial or full-length target cDNA.
Heidecker and Messing, in Meth. in Enz. 154, 28-41 (1987), describe extending the 3' ends of a linearized pUC plasmid with thymidine residues. The oligo(dT) tails are annealed to the poly(A) tails of mRNA and are used to prime cDNA synthesis, thus covalently linking the cDNA to the vector DNA. The plasmid-cDNA molecules are extended with oligo(dG) tails, alkali denatured and sized on alkaline sucrose gradients (thereby also removing the RNA and denaturing the vector). An excess of oligo(dC)-tailed pUC plasmid is added. The vector strands are reannealed, the oligo(dC) tail of the second strand vector serves as primer for synthesis of the second strand of the cDNA insert after which the recombination plasmids are transfected into E. coli. This method also requires a large amount of screening of plasmid vectors in search of vectors containing a partial or full-length target cDNA.
In Coleclough, Meth. In Enz. 154, 64-83 (1987), single stranded cDNA is directly inserted into double stranded cloning vehicles with synthetic oligonucleotides called primer-restriction end adapters, which serve both as primers for polymerase reactions and as ligation substrates. This method also requires a large amount of screening of plasmid vectors in search of vectors containing a partial or full-length target cDNA.
Another method of obtaining full length cDNA clones is the "RACE" protocol, described in Frohman et al., Proc. Natl. Acad. Sci. USA 85, 8998-9002 (1988). The RACE method obtains cDNA clones from rare transcripts, that is, those having a low-abundance of mRNAs. An mRNA selection step is required. Then, cDNA clones are generated by using a modified version of the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the unknown 3' or 5' end. The method requires a single primer which must be a short stretch of sequence within the mRNA to be cloned.
In Joyce et al., Proc. Natl. Acad. Sci. USA 80, 1830-1834 (1983), mass production of the Klenow fragment by directed expression during cloning is described. However, this method also requires a large amount of screening for expression of the lacZ gene, screening by an alkaline lysis procedure, assays for .beta.-galactosidase activity and characterization of fusion junctions by DNA sequence analysis.